PROJECT SUMMARY Tolerance of kidney allografts has been achieved in nonhuman primates (NHPs) and in humans using a combination of nonmyeloablative conditioning and donor bone marrow transplantation (DMBT) that results in transient donor chimerism. However, mixed chimerism protocols that achieve long term tolerance of kidney allografts in NHPs fail to induce tolerance in recipients of heart allografts. It is well known that some organs, such as kidney and liver, are tolerance-prone while others, such as heart and lung, are tolerance-resistant. In earlier studies using miniature swine, we took advantage of the tolerogenicity of kidney allografts and, through donor kidney cotransplantation, achieved long-term stable tolerance of heart allografts which, if transplanted alone, would have rejected acutely. Mechanistic studies in swine showed that kidney allografts were able to confer tolerance upon co-transplanted hearts by promoting the expansion of regulatory T cells (Tregs). We have now extrapolated those findings to NHPs. By combining a mixed chimerism protocol that induces transient donor chimerism with donor kidney cotransplantation, we have succeeded in achieving long-term, stable tolerance of MHC mismatched heart allografts. These results are the first to demonstrate tolerance induction in NHP heart allograft recipients and the first to show that unresponsiveness to a tolerant-resistant organ can be achieved without durable donor chimerism. Based on preliminary results, we hypothesize that amplifying the contributions of host Tregs in NHP heart allograft recipients undergoing mixed chimerism conditioning will achieve long-term, stable tolerance without the need for kidney cotransplantation or durable chimerism. To test this hypothesis, we will first study the characteristics of Tregs generated by kidney cotransplantation and attempt to identify the cells or cell products within the kidney allograft capable of promoting those Tregs. These results will be applied to our subsequent studies aimed at finding a clinically relevant strategy of Treg amplification that effectively substitutes for simultaneous donor kidney transplantation. Our goal is to develop a Treg-amplified, mixed chimerism protocol that can be rapidly translated to human heart transplant recipients. Of note, results from Project 2, which will use novel but related strategies to achieve tolerance of stringent lung allografts, and Project 3, which will use humanized mice to evaluate novel Treg-stabilizing agents will guide the ongoing design of these experiments.